This is in the area of the prevention, diagnosis, and treatment of latent viral infections, such as infections with DNA viruses like Epstein-Barr virus.
Epstein-Barr virus has been known for more than three decades. Epstein-Barr has been associated with cancer and several autoimmune diseases. Since there is evidence implicating Epstein-Barr virus in infectious mononucleosis, B cell lymphoma (in immunocompromised hosts), Burkitt's lymphoma, nasopharyngeal carcinoma, and some cases of Hodgkin's lymphoma, there have been efforts to provide a vaccine against Epstein-Barr virus (Morgan, A. J., et al. J. Med. Virol. 29:74-78 (1989); Morgan, A. J. Vaccine 10:563-571 (1992); Morgan, A. J. Development of Epstein-Barr Virus Vaccines (R. G. Landes Company, Austin, Tex. (1995 by Springer-Verlag, Heidleberg, Germany)); Krause, P. R. & Strauss, S. E. Infect. Dis. Clin. N. A. 13:61-81 (1999)). Recombinant vectors expressing gp340/220 in a bovine papillomavirus vector or in an adenovirus vector protected five of six cottontop tamarins from lymphomas that otherwise occur after infection with Epstein-Barr virus (Finerty, S., et al. J. Gen. Virol. 73:449-453 (1992)). A subunit of the gp340/200 in alum only protected three of five cotton top tamarins from lymphomas (Finerty, S., et al. Vaccine 12:1180-1184 (1994)), suggesting that this strategy might not be especially effective. A trial of an Epstein-Barr virus vaccine of gp340/220 in a Vaccinia virus vector has been reported from China and failed to protect a third ofthose immunized from infection (Gu, S. et al. Dev. Biol. Stand. 84:171-177 (1995)), consistent with the primate data. Khanna, et al., J. Immunol. 162:3063-3069 (1999) reports on the potential use of gp350/gp85 CTL epitopes in vaccine design to protect against EBV. However, as they note, evidence of neutralizing antibodies does not always correlate with protection against infection. Their research is focused on structural antigens, gp350 and gp85, from which they synthesized specific peptides to use as vaccines. They observed higher CTL reactivity to lytic antigens as compared to a latent antigen, LMP-1. Jackman, et al., Vaccine 17:660-668 (1999) also reported on studies using gp350. Gu, et al., Dev. Biol. Stand. 84:171-177 (1995) reports on a clinical trial based on live recombinant vaccinia virus expression gp350 as an immunogen, but there were safety concerns.
Another method of vaccine development has been considered. Cytotoxic T cell epitopes have been mapped for several different EBV antigens in persons of specific HLA haplotypes. For example, trials are underway to evaluate the usefulness of immunization with an EBNA-3 peptide which is a dominant CTL epitope in HLA-B8 restricted persons (Schmidt, et al. Proc. Natl. Acad. Sci. USA 88:9478-82 (1991)). Efficacy of these vaccines is currently unknown.
A variety of additional therapies against Epstein-Barr virus have been attempted. These include inducing the lytic cycle in cells latently infected by virus (Gutierrez, M. I., et al. Cancer Res. 56:969-972 (1996)). Patients with the Epstein-Barr virus related lymphomatoid granulomatosis have been treated with interferon-alpha 2b (Wilson, W. H., et al. Blood 87:4531-4537 (1996)). Cycloheximide and acycloguanosine have been demonstrated to be useful in vitro (Ishii, H. H., et al. Immunol. Cell Biol. 73:463-468 (1995)); however, only limited further evaluation of this therapy has proceeded due to the limited clinical benefit of acycloguanosine in primary EBV infection.
Therapy with a T cell line has been attempted (Kimura, H. et al. Clin. Exp. Immunol. 103:192-298 (1996)), as has adoptive transfer of gene-modified virus-specific T lymphocytes (Heslop, H. E. et al. Nature Med. 2:551-555 (1996)). Data available do not appear to particularly support the use of acyclovir for Epstein-Barr virus infections (Wagstaff, A. J., et al. Drugs 47:153-205 (1994)), though FK506 (a relative of cyclosporine) may have some benefit (Singli, N., et al. Digestive Dis. Sci. 39:15-18 (1994)). Monoclonal antibodies have been used to treat the virus-induced lymphoproliferative syndrome (Lazarovots, A. l., et al. Clin. Invest. Med. 17:621-625 (1994)) with modest early success.
Therapeutic strategies directed against the latent phase of the viral infection have been considered. Among these is the consideration of the use of T cell epitopes of particular HLA-restricted cytotoxic T cells (e.g. see Morgan, A. J. Development of Epstein-Barr Virus Vaccines (R. G. Landes Company, Austin, Texas (1995 by Springer-Verlag, Heidleberg, Germany), pp 109-115; or Bames J. Pharma Weekly 1:11 (1995)). The latent membrane proteins have not been shown to be accessible on the outside of the cell.
Epstein-Barr virus is most similar to the other members of the Herpesviridae family. Although only gamma herpesvirus which infects humans, all human herpesviruses have linear double-stranded DNA, an icosadeltahedral capsid, a tegument which surrounds the capsid and an envelope containing viral glycoprotein spikes on its surface. All human herpesviruses remain with and survive in the host after primary infection.
Cytomegalovirus, in a manner similar to Epstein-Barr virus, is able to establish latency in peripheral immune cells; however, the exact cellular location is still in dispute (Mocarski, E. S. “Cytomegaloviruses and their replication” Fields Virology, third edition (eds. B. N. Fields, et al., Lippincott-Raven (1996)), 2447-2480). Human CMV has been treated with a host of antiviral agents, including leukocyte interferon, interferon stimulators, transfer factor, acyclovir and nucleoside inhibitors, as well as combination therapy with interferon and ara-A (reviewed by Alford, C. A. Antiviral agents and viral diseases of man 2nd ed. New York: Raven Press; 433-86 (1984); Ho, M. Cytomegalovirus. biology and infection: current topics in infectious disease New York: Plenum Press; 105-18 (1982)). Very little to no clinical benefit has been demonstrated with these therapies and overwhelming toxicities are present (Alford, C. A. Antiviral agents and viral diseases of man 2nd ed. New York: Raven Press; 433-86 (1984)).
Gancyclovir (through its nucleic acid chain-terminating activity) and foscarnet (through inhibition of viral DNA polymerase directly) have both been used with success (Snoeck, R., Neyts, J., De Clerq, E. Multidisciplinary approach to understanding cytomegalovinis disease Amsterdam: Excerpta Medica: 1993, 269-78). Both of these drugs have been used with some success in the prophylaxis of invasive CMV in the post-transplant setting. However, drug toxicity and no evident decrease in overall mortality limits their standard use for all patients (Goodrich, J. M, et al. Ann. Intern. Med. 118:173-8 (1993); Reusser P, et al. J. Infect. Dis. 166:473-79 (1992)).
Passive immunoprophylaxis remains very controversial. Newer drugs targeting the CMV protease and DNA processivity activity are under development (Digard, P., Chow, C. S., Pirrit, L., Coen, D. M. J. Virol. 67:1159-68 (1993); Ertl, P. F., Powell, K. L. J. Virol. 66:4126-33 (1992)).
Vaccine development for CMV has met with only limited success. Initial studies were performed using an attenuated laboratory strain of CMV (Elek, S. D., Stern, H. Lancet 1:1-5 (1974); Plotkin, S. A., et al. Infect. Immun. 12:521-27 (1975)). These British studies showed limited immunity which decreased with time (Plotkin, S. A. , et al. J. Infect. Dis. 159:860-65 (1989)). Safety issues concerning immunization of slightly immunocompromized subjects and women of childbearing age have also limited excitement over these studies. Subunit vaccines for CMV prevention are also under consideration. Early studies with the major envelope glycoprotein, gB, are under way (Plotkin, S. A., et al. Rev. Infect. Dis. 12:827-38 (1990); Spaete, R. R., Transplant Proc. 23:90-96 (1991)) and show some early promise.
Human alphaherpesviruses, including herpes simplix-1, herpes simplex-2, varicella-zoster and human herpesvirus 8, primarily establish latency in the sensory ganglia. Substantial evidence is present for the chemotherapy of the herpes-simplex viruses with specific anti-viral therapy, such as acyclovir, gancyclovir and forcarnet (reviewed Rickinson, A. B., Kieff E., “Epstein-Barr virus”, Fields Virology 2397-2446 (1996)). Numerous vaccines for HSV-1 and HSV-2 have been developed with poor success (reviewed by Whitley, R. J. “Herpes Simplex viruses”, Field's Virology 2297-2330 (1996)). Two new approaches are of interest. One is based upon the production of adequate amounts of recombinant HSV-2 glycoproteins B or D to be used either separately or together as a subunit vaccine (Ashley, R., Mertz, G. J., Corey, L. J. Virol. 61:253-8 (1987); Zarling, J. M., et al. J. Virol. 62:4481-85 (1988)). Concerns with these studies include the use of Freund's adjuvant or a lipophilic muramyl tripeptide, neither of which is acceptable human adjuvants. The second method is to genetically engineer a live, attenuated recombinant HSV that combines type 1 and type 2 genomes without the putative neurovirulence sequences (Meignier, B., Longnecker, R., Roizman, B. J. Infect. Dis. 162:313-21 (1990)).
Varicella zoster also establishes latency in sensory neurons. This virus in its typical primary infection of the juvenile host is only treated with supportive measures with full recovery usually within one week. However, life-threatening primary and recurrent infections are encountered in immunocompromized hosts. These overwhelming infections are usually treated with acyclovir. Gancyclovir and famcylovir are used for treatment of resistant strains. A live, attenuated varicella vaccine is in common use; however, long-term efficacy studies for this vaccine are still pending.
The human herpesvirus-8 is the other alpha herpesvirus. Only recently identified and characterized, neither therapy trials nor vaccine development has yet begun.
The problem within latent viral infections is the ability to identify, target, and treat cells harboring latent virus. For example, the Epstein-Barr virus produces certain proteins during the latent stage of its life cycle. These proteins have not been shown to be antigenic in an expression system that would enable immune system recognition. One of these proteins is LMP-2A. Antibodies have been raised against LPM-2A and immunofluoresence used to show that LMP-2A is located in or near the membrane (Longnecker, R. and Kieff, E., J. Virol. 64:3219-2326 (1990)). These workers established that their antiserum bound to LMP-2A isolated from the cell, but it was not established that the extracellular part of LMP-2A was antigenic, i.e., could elicit antibody production or was accessible to be bound by antibody (or any other external ligand). In fact, these workers concluded that their antibodies recognized the intracellular portion of LMP-2A because the antibodies were LMP-2A specific and it is the LMP-2A intracellular region which differentiates LMP-2A from LMP-2B. (Actually, these two Epstein-Barr virus proteins are in large part identical and appear to vary because splice junction variation gives LMP-2A a longer amino terminal cytoplasmic tail than is found in LMP-2B.)
It is therefore an object of the present invention to provide methods to target cells that are infected with a virus, such as a DNA virus like a herpes virus, in the latent stage of its life cycle.
It is a further object of the present invention to provide strategies to target cells containing Epstein-Barr virus in a latent state.
It is another object of the present invention to provide strategies to treat or prevent diseases linked to these latent viruses.
It is another object of the present invention to provide vaccines based upon the structure of proteins expressed during the latent life cycle of a virus, such as Epstein-Barr virus.
It is a further object of the present invention to provide methods for the development of therapeutics to treat diseases linked to Epstein-Barr virus by targeting cells latently infected with Epstein-Barr virus.
It is a further object of this invention to provide diagnostics which will identify people with latent viral infections, such as infections with Epstein-Barr virus.
It is a further object of this invention to provide diagnostic tests which will help distinguish those with a disease from those without a disease by differences in the immune responses to a DNA virus, such as Epstein-Barr virus.